Integrated Transcriptome Analysis Reveals the Impact of Photodynamic Therapy on Cerebrovascular Endothelial Cells

Blood vessels in the brain tissue form a compact vessel structure and play an essential role in maintaining the homeostasis of the neurovascular system. The low dosage of photodynamic intervention (PDT) significantly affects the expression of cellular biomarkers. To understand the impact of photodynamic interventions on cerebrovascular endothelial cells, we evaluated the dosage-dependent impact of porfimer sodium-mediated PDT on B.END3 cells using flow cytometer, comet assay, RNA sequencing, and bioinformatics analysis. To examine whether PDT can induce disorder of intracellular organelles, we did not observe any significance damage of DNA and cellular skeleton. Moreover, expression levels of cellular transporters-related genes were significantly altered, implying the drawbacks of PDT on cerebrovascular functions. To address the potential molecular mechanisms of these phenotypes, RNA sequencing and bioinformatics analysis were employed to identify critical genes and pathways among these processes. The gene ontology (GO) analysis and protein-protein interaction (PPI) identified 15 hub genes, highly associated with cellular mitosis process (CDK1, CDC20, MCM5, MCM7, MCM4, CCNA2, AURKB, KIF2C, ESPL1, BUB1B) and DNA replication (POLE2, PLOE, CDC45, CDC6). Gene set enrichment analysis (GSEA) reveals that TNF-α/NF-κB and KRAS pathways may play a critical role in regulating expression levels of transporter-related genes. To further perform qRT-PCR assays, we find that TNF-α/NF-κB and KRAS pathways were substantially up-regulated, consistent with GSEA analysis. The current findings suggested that a low dosage of PDT intervention may be detrimental to the homeostasis of blood-brain barrier (BBB) by inducing the inflammatory response and affecting the expression of surface biomarkers.

Claudin-5 Affects Endothelial Autophagy in Response to Early Hypoxia

Hypoxic injury to cerebrovascular endothelial cells (ECs) after stroke leads to blood-brain barrier (BBB) dysfunction, which is commonly associated with disruptions of endothelial tight junctions (TJs) and increased permeability. Therefore, maintaining the structural integrity and proper function of the BBB is essential for the homeostasis and physiological function of the central nervous system (CNS). Our previous study revealed that autophagy functions on protecting the BBB by regulating the dynamics of Claudin-5, the essential TJ protein, under short-term starvation or hypoxia conditions. Here, we show that in zebrafish and in vitro cells, loss of membranous Claudin-5 conversely determine the occurrence of hypoxia-induced autophagy in cerebrovascular ECs. Absence of endothelial Claudin-5 could partly attenuate endothelial cell apoptosis caused by short-term hypoxic injury. Mechanism studies revealed that under hypoxic conditions, the existence of membranous Claudin-5 affects the stimulation of hypoxia inducible factor 1 subunit alpha (HIF-1a) and the inducible nitric oxide synthase (iNOS), which are responsible for the translocation of and endocytosis of caveole-packaged Claudin-5 into cytosol. Meanwhile, loss of Claudin-5 affects the generation of reactive oxygen species (ROS) and the downstream expression of BCL2/adenovirus E1B 19kDa protein interacting protein 3 (Bnip3). These together suppress the endothelial autophagy under hypoxia. This finding provides a theoretical basis for clarifying the mechanism of hypoxia-induced BBB injury and its potential protection mechanisms.

NEK1-mediated retromer trafficking promotes blood–brain barrier integrity by regulating glucose metabolism and RIPK1 activation

Loss-of-function mutations in NEK1 gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS. Here we show that NEK1 regulates retromer-mediated endosomal trafficking by phosphorylating VPS26B. NEK1 deficiency disrupts endosomal trafficking of plasma membrane proteins and cerebral proteome homeostasis to promote mitochondrial and lysosomal dysfunction and aggregation of α-synuclein. The metabolic and proteomic defects of NEK1 deficiency disrupts the integrity of blood–brain barrier (BBB) by promoting lysosomal degradation of A20, a key modulator of RIPK1, thus sensitizing cerebrovascular endothelial cells to RIPK1-dependent apoptosis and necroptosis. Genetic inactivation of RIPK1 or metabolic rescue with ketogenic diet can prevent postnatal lethality and BBB damage in NEK1 deficient mice. Inhibition of RIPK1 reduces neuroinflammation and aggregation of α-synuclein in the brains of NEK1 deficient mice. Our study identifies a molecular mechanism by which retromer trafficking and metabolism regulates cerebrovascular integrity, cerebral proteome homeostasis and RIPK1-mediated neuroinflammation.

cAMP Compartmentalization in Cerebrovascular Endothelial Cells: New Therapeutic Opportunities in Alzheimer’s Disease

The vascular hypothesis used to explain the pathophysiology of Alzheimer’s disease (AD) suggests that a dysfunction of the cerebral microvasculature could be the beginning of alterations that ultimately leads to neuronal damage, and an abnormal increase of the blood–brain barrier (BBB) permeability plays a prominent role in this process. It is generally accepted that, in physiological conditions, cyclic AMP (cAMP) plays a key role in maintaining BBB permeability by regulating the formation of tight junctions between endothelial cells of the brain microvasculature. It is also known that intracellular cAMP signaling is highly compartmentalized into small nanodomains and localized cAMP changes are sufficient at modifying the permeability of the endothelial barrier. This spatial and temporal distribution is maintained by the enzymes involved in cAMP synthesis and degradation, by the location of its effectors, and by the existence of anchor proteins, as well as by buffers or different cytoplasm viscosities and intracellular structures limiting its diffusion. This review compiles current knowledge on the influence of cAMP compartmentalization on the endothelial barrier and, more specifically, on the BBB, laying the foundation for a new therapeutic approach in the treatment of AD.

Posterior Reversible Leukoencephalopathy Syndrome in a Patient after Acute COVID-19 Infection

The SARS-CoV-2 infection affects numerous organs, including the central nervous system. The neuroinvasive abilities and neuroinflammation may lead to short- and long-term neurological manifestations. Among neurological disorders associated with SARS-CoV-2 infection, posterior reversible encephalopathy syndrome (PRES) has been described in a few case-based observational studies during the acute phase of COVID-19 hospitalization. We present a case of a patient who developed seizures and PRES after recovering from an acute severe COVID-19 infection.A 90-year-old African American female with multiple comorbidities and a severe COVID-19 infection was discharged home in stable condition after two weeks of hospitalization. A week later, she developed new-onset generalized tonic-clonic seizures requiring readmission to the hospital. The patient’s clinical course and brain imaging supported PRES. Her mentation returned to baseline with supportive care and anticonvulsant treatment. Follow-up brain MRI four months later demonstrated resolution of FLAIR signal abnormalities confirming PRES. SARS-CoV-2 insult on the cerebrovascular endothelial cells likely continued and despite the clinical recovery eventually resulted in PRES. We believe that this is the first case describing the presentation of PRES after recovery from severe acute COVID-19 infection.

Auraptene Enhances Junction Assembly in Cerebrovascular Endothelial Cells by Promoting Resilience to Mitochondrial Stress through Activation of Antioxidant Enzymes and mtUPR

Junctional proteins in cerebrovascular endothelial cells are essential for maintaining the barrier function of the blood-brain barrier (BBB), thus protecting the brain from the infiltration of pathogens. The present study showed that the potential therapeutic natural compound auraptene (AUR) enhances junction assembly in cerebrovascular endothelial cells by inducing antioxidant enzymes and the mitochondrial unfolded protein response (mtUPR). Treatment of mouse cerebrovascular endothelial cells with AUR enhanced the expression of junctional proteins, such as occludin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin), by increasing the levels of mRNA encoding antioxidant enzymes. AUR treatment also resulted in the depolarization of mitochondrial membrane potential and activation of mtUPR. The ability of AUR to protect against ischemic conditions was further assessed using cells deprived of oxygen and glucose. Pretreatment of these cells with AUR protected against damage to junctional proteins, including occludin, claudin-5, ZO-1 and VE-cadherin, accompanied by a stress resilience response regulated by levels of ATF5, LONP1 and HSP60 mRNAs. Collectively, these results indicate that AUR promotes resilience against oxidative stress and improves junction assembly, suggesting that AUR may help maintain intact barriers in cerebrovascular endothelial cells.

Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

Amyloid β-protein (Aβ) may contribute to worsening of Alzheimer’s disease (AD) through vascular dysfunction, but the actual molecular mechanisms remain controversial. Using ex-vivo blood vessels and primary endothelial cells derived from human brain microvessels, we revealed that patient-derived Aβ assemblies, termed amylospheroids (ASPD), exist on the microvascular surface in patient brains and inhibit vasorelaxation through binding to the α3 subunit of sodium, potassium-ATPase (NAKα3) on endothelial cells. Interestingly, NAKα3 also serves as the toxic target of ASPD in neurons. ASPD elicit neurodegeneration through calcium overload, while ASPD suppress vasorelaxation by inhibiting nitric oxide (NO) production. ASPD-NAKα3 interaction on cerebrovascular endothelial cells disturbs the NO release by inactivating endothelial NO synthase through mitochondrial reactive oxygen species and protein kinase C. The findings suggest that ASPD may dually contribute to neuronal and vascular pathologies through binding to NAKα3. Thus, blocking the ASPD-NAKα3 interaction may be a useful target for AD therapy.

3-Mercaptopyruvate sulfurtransferase/hydrogen sulfide protects cerebral endothelial cells against oxygen-glucose deprivation/reoxygenation-induced injury via mitoprotection and inhibition of the RhoA/ROCK pathway

3-Mercaptopyruvate sulfurtransferase (3-MST) is the major source of hydrogen sulfide (H2S) production in the brain and participates in many physiological and pathological processes. The present study was designed to investigate the role of 3-MST-derived H2S (3-MST/H2S) on oxygen-glucose deprivation/reoxygenation (OGD/R) injury in cerebrovascular endothelial cells (ECs). Using cerebrovascular specimens from patients with acute massive cerebral infarction (MCI), we found abnormal morphology of the endothelium and mitochondria, as well as decreases in H2S and 3-MST levels. In an OGD/R model of ECs, 3-mercaptopyruvate (3-MP) and l-aspartic acid (l-Asp) were used to stimulate or inhibit the production of 3-MST/H2S. The results showed that OGD/R induced significant decreases in H2S and 3-MST levels in both ECs and mitochondria, as well as increases in oxidative stress and mitochondrial energy imbalance. Cellular oxidative stress, destruction of mitochondrial ultrastructure, accumulation of mitochondrial reactive oxygen species (ROS), reduction of mitochondrial adenosine triphosphate (ATP) synthase activity and ATP production, and decreased mitochondrial membrane potential were all significantly ameliorated by 3-MP, whereas they were exacerbated by l-Asp pretreatment. Contrary to the effects of l-Asp, the increase in RhoA activity and expression of ROCK1 and ROCK2 induced by OGD/R were markedly inhibited by 3-MP pretreatment in subcellular fractions without mitochondria and mitochondrial fractions. In addition, 3-MST−/− rat ECs displayed greater oxidative stress than 3-MST+/+ rat ECs after OGD/R injury. These findings suggest that 3-MST/H2S protects ECs against OGD/R-induced injury, which may be related to preservation of mitochondrial function and inhibition of the RhoA/ROCK pathway.

Pituitary macroadenoma apoplexy in a severe acute respiratory syndrome-coronavirus-2-positive testing: Causal or casual?

Background: In December 2019, in Wuhan, a new virus emerged, causing severe acute respiratory syndrome (SARS) secondary to infection by a type of coronavirus, causing coronavirus disease (COVID-19). The pandemic caused by the new coronavirus has had implications in the central nervous system. COVID-19 is known to be characterized by coagulation activation and endothelial dysfunction, causing ischemic and hemorrhagic vascular syndromes. Case Description: A 27-year-old male patient case with progressive decrease in visual acuity, associated with respiratory symptoms and intense headache. Multilobar infiltrate with a reticulonodular pattern is evident on chest CT scan. Brain CT scan with pituitary macroadenoma apoplexy was shown. SARS-Cov2 was confirmed, and respiratory support initiated. However, the patient died shortly afterward, secondary to pulmonary complications. Conclusion: The angiotensin-converting enzyme (ACE) II receptor is expressed in circumventricular organs and in cerebrovascular endothelial cells, which play a role in vascular autoregulation and cerebral blood flow. For this reason, is rational the hypothesize that brain ACE II could be involved in COVID-19 infection. Underlying mechanisms require further elucidation in the future.